Early life 5-HT signaling influences neurodevelopmental trajectories and altered 5-HT signaling has been implicated in the pathogenesis of numerous stress-related psychiatric disorders. What is not clear is how extracellular 5-HT exerts its effects on nervous system development and whether the critical synthetic source of 5-HT is the brain. In Project 1: Early Brain Serotonin and its Lasting Impact on Neuronal Epigenetic Programming, Evan Deneris seeks to determine whether 5-HT synthesized specifically in hindbrain raphe neurons and secreted during fetal and early postnatal life is an important extracellular signal required for early-life epigenetic programming of serotonergic homeostasis and hypothalamic-pituitary-adrenal (HPA) axis stress circuitry. To investigate this hypothesis, Deneris seeks to apply his recently developed temporally controlled targeting approaches to knock out the gene, tryptophan hydroxylase 2, responsible for synthesis of brain 5-HT. Tph2 will be targeted during fetal and early postnatal life to reduce brain 5-HT synthesis but not synthesis of 5-HT from exogenous sources such as the placental or gut. The targeting of Tph2 at different stages of early life enables Deneris to investigate a series of questions that have been difficult or impossible to address with previous approaches aimed at determining the developmental impact of brain 5 HT synthesis. In Specific Aim 1, Deneris will target Tph2 during fetal life and during the critical early postnatal period to investigate a potential role for 5-HT as an autocrine signal required for homeostatic maintenance of intrinsic 5-HT neuron transcriptional programs, intrinsic 5-HT neuron biochemical and physiological properties and RNA editing patterns. In Specific Aim 2, Deneris seeks to initiate a novel study of the previously unexplored serotonergic epigenome and determine how it impacts stress-related behaviors. Tph2 targeted mice will be used to determine the impact of early-life 5-HT on long lasting programming of histone acetylation/methylation marks and DNA promoter methylation patterns in serotonergic genes and serotonergic histone deacetylase (HDAC) expression. 5-HT neuron-type targeting of HDAC2, an HDAC strongly expressed in developing 5-HT neurons, will be used to determine how alterations in the serotonergic epigenome impacts 5-HT neuron function and stress-related behaviors. In Specific Aim 3, Deneris' team will utilize his powerful 5-HT neuron-type genetic strategies to directly test the long standing hypothesis that 5 HT produced in the brain is a developmental transducer of early life experience and is required to epigenetically program development of the HPA axis and protect against the effects of early life stress.